Subunit vaccines are vaccines which are devoid of intact pathogen cells. These vaccines are usually composed of substantially purified antigens. Such vaccines are generally preferable to compositions which use attenuated or inactivated pathogens. However, many subunit vaccines which include proteins, such as peptide hormones and bacterial and viral antigens, require the help of a carrier protein in order to elicit a strong immune response. This is especially true for small proteins or endogenous substances, such as hormones, which are poorly immunogenic.
The carrier serves to non-specifically stimulate T helper cell activity and to direct the antigen to the antigen presenting cell, where the antigen is processed and presented at the cell surface in the context of molecules of the major histocompatibility complex (MHC).
Several carrier systems have been developed for this purpose. For example, small peptide antigens are often coupled to protein carriers such as keyhole limpet haemocyanin (Bittle, J. L., et al., Nature (1982) 298:30-33), tetanus toxoid (Muller, G., et al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79:569-573), ovalbumin, and sperm whale myoglobin, to produce an immune response. However, carriers may elicit strong immunity not relevant to the peptide antigen and this may inhibit the immune response to the peptide vaccine on secondary immunization (Schutze, M. P., et al, J. Immun. (1985) 135:2319-2322).
Antigen delivery systems have also been based on particulate carriers. For example, preformed particles have been used as platforms onto which antigens can be coupled and incorporated. Systems based on proteosomes (Lowell, G. H., et al., Science (1988) 240:800-802), immune stimulatory complexes (Morein, B., et al., Nature (1984) 308:457-460), and viral particles such as HBsAg (Neurath, A. R., et al., Mol. Immunol. (1989) 26:53-62) and rotavirus inner capsid protein (Redmond, M. J., et al., Mol. Immunol. (1991) 28:269-278) have been developed.
Other carrier systems have been devised using recombinantly produced chimeric proteins that self assemble into particles. For example, the yeast retrotransposon, Ty, encodes a series of proteins that assemble into virus like particles (Ty-VLPs; Kingsman, S. M., and A. J. Kingsman Vacc. (1988) 6:304-306). Foreign genes have been inserted into the TyA gene and expressed in yeast as a fusion protein. The fusion protein retains the capacity to self assemble into particles of uniform size.
Other chimeric protein particles have been examined such as HBsAg, (Valenzuela, P., et al., Bio/Technol. (1985) 3:323-326; U.S. Pat. No. 4,722,840; Delpeyroux, F. N., et al., Science (1986) 233:472-475), Hepatitis B core antigen (Clarke, B. E., et al., Vaccines 88 (Ed. H. Ginsberg, et al., 1988) pp. 127-131), Poliovirus (Burke, K. L., et al., Nature (1988) 332:81-82), and Tobacco Mosaic Virus (Haynes, J. R., et al., Bio/Technol. (1986) 4:637-641). However, these carriers are restricted in their usefulness by virtue of the limited size of the active agent which may be inserted into the structural protein without interfering with particle assembly.
Gene fusions provide a convenient method for the production of chimeric proteins. The expression of chimeric proteins affords an efficient means of linking a carrier protein to a desired antigen.
Pasteurella haemolytica produces a cytotoxin which is a leukotoxin. See, e.g. Gentry et al. Vet. Immunology and Immunopathology (1985) 9:239-250; Shewen, P. E., and Wilkie, B. N. Infect. Immun. (1987) 55:3233-3236. The gene encoding this cytotoxin has been cloned and expressed in bacterial cells. Lo et al. Infect. Immun. (1985) 50:667-671; U.S. Pat. No. 5,055,400. The leukotoxin has been used as an antigen in vaccine formulations to fight shipping fever pneumonia in livestock (See, e.g. U.S. Pat. No. 4,957,739) as well as to produce chimeric molecules for use in vaccines against shipping fever (see, e.g. International Publication No. WO 92/03558, published 5 Mar. 1992; and U.S. Pat. No. 5,028,423). However, the use of leukotoxin as a carrier molecule to increase the immune response of antigens associated therewith has not heretofore been described.